Methods, devices, kits and compositions for detecting roundworm

ABSTRACT

Methods, devices, kits and compositions for detecting the presence or absence of roundworm in a mammalian sample are disclosed herein. The methods, devices, kits and compositions of the present invention may be used to confirm the presence or absence of roundworm in a fecal sample from a mammal that may also be infected with one or more of hookworm, whipworm, and heartworm. Confirmation of the presence or absence of roundworm in the mammal may be made, for example, for the purpose of selecting an optimal course of treating the mammal and/or for the purpose of determining whether the mammal has been rid of the infection after treatment has been initiated.

CROSS REFERENCE

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/128,079, filed May 19, 2008, which is incorporated byreference herein in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compositions, devices, kits and methodsfor the detection of roundworm in mammals. More particularly, thepresent invention relates to polypeptides and polypeptide compositions,antibodies and antibody compositions, devices, kits, and methods fordetecting the presence or absence of roundworm antigen in a sample froma mammal that may also include one or more of hookworm, whipworm, andheartworm antigen.

2. Description of the Prior Art

Adult roundworms live in the small intestine and lay eggs that pass outin the feces. In the environment, infective larvae remain within theeggs and develop into an infective stage after approximately three weeksat optimal temperatures. The infective eggs enter a host by ingestionand hatch in the small intestine. In dogs less than five weeks of age,larvae migrate through the tissue and into the bloodstream beforeeventually reaching the lung and trachea where additional developmentoccurs. The host coughs up and swallows the larvae, which molt intoadults that reside in small intestine. Larvae that hatch within dogsgreater than five weeks of age or within other animals, includinghumans, are capable of traveling to a wide range of tissues includingthe liver, lungs, heart, brain, and skeletal muscle. These larvaesubsequently arrest their development and encyst in the tissue of thehost. In pregnant and lactating dogs, encysted larvae can becomereactivated and cause intestinal infection in the mother, migrate to theuterus and directly infect the fetus through the placenta, or migrate tothe mammary tissue and infect nursing animals. Parasitic roundwormscause disease not only in their animal hosts, but are also theetiological agents of larval migrans syndrome as well as severeenteritis and allergic reactions in humans, which occurs after ingestionof infectious eggs from the environment or ingestion of larvae foundwithin liver, meat or other tissues of paratenic hosts.

Intestinal roundworm infection is common in animals and, if leftuntreated, can cause serious disease and even death. Although it isrelatively easy to diagnose a roundworm-infected animal as having aparasitic worm (helminth) infection of some type, it is significantlymore difficult to identify roundworm, specifically, as the causativeworm. This is a problem because roundworm infections are best treatedwhen the infected animal's caregiver has knowledge that roundworm is thespecific source of the infection. In addition, humans who may come incontact with the infested animal or its excretions may be advised totake precautions against acquiring the parasite. In this context, it isimportant to determine the worm species with high specificity, as somehelminths, such as roundworms and hookworms, can cause significantdisease (e.g., larva migrans) in humans, while it is generally acceptedthat whipworm does not play a zoonotic role of importance in humans.

Current methods for diagnosis of roundworm infections primarily involvemicroscopic examination of fecal samples, either directly in fecalsmears or following concentration of ova by flotation in density media.Despite this procedure's high adoption, the method has significantshortcomings. These microscopic methods are time consuming, areunpleasant, require specialized equipment and can have low specificityand sensitivity [Dryden et al., 2005, Vet Therap. 6(1), 15-28]. Inaddition, the accuracy of results of these methods is highly dependentupon the skill and expertise of the operator.

Stool handling is disagreeable and hazardous. Sanitary and inoffensiveprocedures for processing stool are awkward and often complex. Suchprocedures may include weighing, centrifuging and storing, and aredifficult except in a clinical laboratory equipped with a suitableapparatus, protective equipment, and a skilled technician. Therefore,any reduction in the number of steps required to perform a fecal testand any reduction in contact between test operator and the test materialis desirable. Clinical laboratories have been using the immunoassaymethods for the detection of various viruses, bacteria and non-helminthparasites and organisms in feces. However, there remains a need for asimple immunoassay method for the detection of a parasitic worminfection, and roundworm infection in particular in feces, whole bloodor in serum.

SUMMARY OF THE INVENTION

In one aspect, the invention includes antibodies that specifically bindto a polypeptide including all or an antigenic portion of the amino acidsequence that corresponds to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, as listed herein, or to a polypeptide including asequence that is a conservative variant of one of those sequences. In afurther aspect, the antibodies specifically bind to antigen fromroundworm infested mammals, but do not specifically bind antigen frommammals infected with hookworm, heartworm and/or whipworm.

In another aspect, the invention includes antibodies that are obtainedby immunization with the polypeptide including all or an antigenicportion of the amino acid sequence that corresponds to SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, or with a polypeptideincluding a sequence that is a conservative variant of one of thosesequences.

In yet another aspect, the invention provides a device for detecting thepresence or absence of roundworm antigens from a sample; the devicecomprising a solid support, wherein the solid support has immobilizedthereon one or more antibodies that are capable of specifically bindingto a polypeptide that has an amino acid sequence that corresponds to SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or anantigenic portion thereof. The device, may be, but is not limited tobeing, for example, an ELISA device, such as a lateral flow immunoassaydevice or microtiterplate device. Mammalian samples that may be testedfor roundworm by the device include, but are not limited to being,feces, whole blood, serum, mammary milk and whole tissue, such as tissuefrom mammary gland, intestine, liver, heart, lung, esophagus, brain,muscle, and eye, for example. The device further may include, but neednot include, one or more reagents for the detection of one or more ofthe group consisting of: one or more non-roundworm worm parasites, oneor more non-worm parasites, one or more viruses, one or more fungi, andone or more bacteria.

In yet another aspect, the invention provides a method of detecting thepresence or absence of roundworm, such as Toxocara canis (T. canis),Toxocara cati (T. cati), Toxocara vitulorum (T. vitulorum), Toxascarisleonina (T. leonina), Baylisascaris procyonis (B. procyonis), Ascaridiagalli (A. galli), Parascaris equorum (P. equorum), Ascaris suum (A.suum), or Ascaris lumbricoides (A. lumbricoides), Anisakis simplex (A.simplex), or Pseudoterranova decipiens (P. decipiens), for example, in asample. The sample can be obtained from a mammal, such as a canine,feline, porcine, bovine, cetacean, pinniped or human. In one aspect, themethod is carried out to test a fecal sample for roundworm coproantigen.The method, however, is not limited to being carried out to test a fecalsample. In addition to feces, the sample therefore may be, but is notlimited to being whole blood, serum, mammary milk and whole tissue, suchas tissue from mammary gland, intestine, liver, heart, lung, esophagus,brain, muscle, and eye, for example. Steps of the method includecontacting the sample with one or more of the antibodies of theinvention; forming antibody polypeptide complexes in the presence of thepolypeptides if any, in the sample; and detecting the presence orabsence of the antibody-polypeptide complexes, if any. The methodfurther may include one or more of the optional steps of diagnosing themammal as either having or not having a roundworm infection anddetermining whether a nucleic acid from roundworm is present in the samesample that was contacted with the antibodies for the purpose ofdetecting the presence or absence of roundworm or in some other samplefrom the mammal. The method may also be used to test for environmentalcontamination with roundworm. Environmental samples that may be testedfor roundworm by the device include, but are not limited to soil,decomposing material, or fecal matter from residential settingsincluding yards, gardens, sand boxes, and playgrounds. Testing locationsmay also include parks, beaches, forests, farms, or other locationsexposed to fecal material from dogs, cats, or other mammalian hosts ofroundworms. Feces from indoor and outdoor litter boxes may also betested.

In yet another aspect, the present invention includes a kit for carryingout one or more steps of the method of the invention. The kit mayoptionally include, for example, the device and one or more of thecompositions of the present invention and instructions for carrying outthe method of the present invention. The kit may further optionallyinclude, for example, one or more indicator reagents, one or moreantibody labeling compounds, one or more antibodies, one or more antigencapture reagents, one or more inhibitors, and one or more wash reagentsto be used as part of the device and/or to be used in carrying out themethod.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of an 865-nucleotide cDNA sequencefrom whole adult Toxocara canis. (SEQ ID NO:1).

FIG. 2 shows the nucleotide sequence of an 632-nucleotide cDNA sequencefrom whole adult Toxocara cati. (SEQ ID NO:2).

FIG. 3 shows the amino acid sequence (SEQ ID NO:3) of a large openreading frame (ORF) of SEQ ID NO: 1. The stop codon is indicated by *.

FIG. 4 shows the amino acid sequence (SEQ ID NO:4) of a large ORF of SEQID NO:2. The stop codon is indicated by *.

FIG. 5 shows a comparison alignment of SEQ ID NO:4 and SEQ ID NO:5. Theconsensus sequence of SEQ ID NO:4 and SEQ ID NO:5 is shown as SEQ IDNO:7.

FIG. 6A shows a multi-well plate device of the present invention.

FIG. 6B shows a close up of a single well of the plate of FIG. 6A with aspecific antibody of the present invention immobilized thereto.

FIG. 7 shows a graph of optical density (OD) values obtained from fecalsamples from roundworm-infected canines by following the method of thepresent invention in a first Example.

FIG. 8 shows a graph of OD values obtained from fecal samples fromcanines infected with either roundworm, hookworm or whipworm byfollowing the method of the present invention in a second Example.

FIG. 9 shows a graph of OD values obtained from fecal samples fromcanines infected with heartworm by following the method of the presentinvention in the second Example.

FIG. 10 shows a microtiter plate in which an ELISA assay was carried outusing fecal samples from canines infected with either roundworm,hookworm, whipworm or heartworm by following the method of the presentinvention in a third Example.

FIG. 11 shows a graph of OD values obtained from fecal samples from aset of canines when those canines had a roundworm infection, and fromfecal samples from those canines after they had been rid of roundworm,by following the method of the present invention in a fourth Example.

FIG. 12 shows a graph of OD values obtained from fecal samples fromroundworm-infected felines by following the method of the presentinvention in a fifth Example.

FIG. 13 shows a first graph of OD values obtained from fecal samplesfrom a set of felines when those felines had a roundworm infection, andfrom fecal samples from those felines after they had been rid ofroundworm, by following the method of the present invention in a sixthExample.

FIG. 14 shows a second graph of OD values obtained from fecal samplesfrom a control set of felines that were not infected with roundworm byfollowing the method of the present invention in the sixth Example.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

I. Introduction

The present invention is generally directed to methods, devices, kitsand compositions for detecting roundworm in a sample obtained from amammal. The present invention relates to roundworm antigens fromToxocara, such as Toxocara canis or Toxocara cati, for example. Inparticular, the present invention relates to Toxocara polypeptides andconservative variants thereof, polynucleotides that encode thosepolypeptides and oligonucleotides that specifically bind to thosepolynucleotides, antibodies that are raised against and thatspecifically bind those polypeptides, and methods, devices and kits fordetecting roundworm, such as Toxocara, Toxascaris, Baylisascaris,Ascaridia, Parascaris, and Ascaris, Anisakis, or Pseudoterranova,including T. canis, T. cati, T. vitulorum, T. leonina B. procyonis, A.galli, P. equorum, A. lumbricoides, A. suum, A. simplex, or P.decipiens, for example.

The present invention provides a superior alternative to these existingmicroscopic inspection techniques. This is true because the presentinvention provides compositions, devices, kits and methods for detectingthe presence or absence of roundworm in a sample from a mammal that: (1)are both easy to use and yield consistently reliable results; (2) allowfor the absence or presence of roundworm in a mammal to be confirmedregardless of whether that mammal is infected with hookworm, whipworm,and/or heartworm; and (3) can detect roundworm prior to the time thatroundworm ova first appear in the infected host's feces.

The present invention is based in part on the discovery of unexpectedproperties of compositions of the present invention. Specifically, itwas determined that an antibody of the present invention raised againsta polypeptide of the present invention can be used to capture and detectroundworm antigens in a mammal, even when the mammal is also infested byone or more of hookworm, whipworm and heartworm. This specificity forroundworm is surprising because roundworms, whipworms, hookworms andheartworms all are related nematodes, and an antibody raised against aprotein isolated from any one of these worms would be expected tocrossreact with one or more of the other worms, host antigens, or otherhost components.

It was further determined that this antibody can be used to capture anddetect roundworm antigens in a mammal as early as 17 days after themammal is first infected with roundworm. This ability to detectroundworm so soon after infection, and before the appearance of anyroundworm ova in the feces of the infected mammal, is surprising becauseroundworm ova generally do not appear in the feces of an infective hostuntil about five-to-eight weeks after the host becomes infected.

The present invention therefore includes methods, devices, compositionsand kits that use antibodies and/or fragments thereof to specificallycapture and detect roundworm antigens in a mammal that may also beinfested by one or more of whipworm, hookworm and heartworm. The abilityof the present invention to detect and diagnose roundworm even when oneor more other worm types are also present allows the mammal's caregiverthe opportunity to optimally select a treatment for ridding theroundworm from the mammal. Further, the ability of the present inventionto, in some cases, detect roundworm as early as 17 days after the mammalis first infected provides the possibility that the caregiver may beginsuch treatment before the mammal becomes severely sickened by theroundworm. An intervention prior to appearance of ova in the feces wouldalso greatly reduce or eliminate the possibility that the infestation isspread to other animals or humans.

II. Definitions and Uses of Terms

The term “compositions of the invention” refers to all of the nucleicacids, polypeptides, antibodies, and mixtures that include one or moreof those nucleic acids, polypeptides, and antibodies and one or moreother compounds, that can be used to detect the presence or absence ofroundworm in a sample obtained from a mammal by carrying out the methodof the present invention that are explicitly described, implicitlyencompassed or otherwise disclosed herein.

“A sample from a mammal” in which roundworm can be detected by thepresent invention includes all bodily components and extracts thereof,such as any fluid, solid, cell or tissue, that are capable of containingroundworm antigen. Exemplary samples therefore include, but are notlimited to being, feces, milk, whole blood and portions thereof,including serum, and further include tissue extracts, including tissuefrom mammary gland, intestine, liver, heart, lung, esophagus, brain,muscle, and eye, for example. The sample may be taken directly from themammal or the sample may be taken from anything that has contacted themammal. For example, the sample may be fresh or decaying fecal droppingsfrom the mammal. As another example, the sample may include soil, dirt,sand, plant material, or any other material that may be mixed withbodily components that may be left behind by a mammal, such as feces,for example. No matter the origin or the content of the sample, thissample sometimes is referred to herein as the “mammalian sample”, the“test sample” or the “sample under test”.

As used herein, “nucleic acid” is synonymous with, and therefore is usedinterchangeably with, “gene”, “DNA”, “cDNA”, “EST”, “polynucleotide”,“oligonucleotide”, “polynucleic acid”, “RNA” and “mRNA”. A nucleic acidmay be in double-stranded form or it may be in single-stranded form.Further, a nucleic acid is either naturally isolated, such as from awhole roundworm or a portion thereof, for example, or it is artificiallysynthesized, either in a recombinant host organism or by any otherartificial means known to the skilled artisan, such as by employing aPCR-based technique, by creating a transgenic organism that synthesizesthe nucleic acid, by using a DNA synthesizing machine, or by any anothermolecular-based technique, for example.

“Polypeptide”, “peptide” and “protein” are synonymous terms that areused interchangeably herein to refer to a polymer of amino acidresidues. A polypeptide, peptide and protein of the present inventionmay be either naturally isolated, such as from a whole roundworm or froma portion of roundworm, for example, or artificially synthesized, eitherin a recombinant host organism or by any other artificial means known tothe skilled artisan.

The term “antibody” or “antibody of the present invention” refers to anyantibody that is able to specifically bind to one or more roundwormantigens, but not to any antigen from hookworm, whipworm or heartworm.The antibodies of the present invention may be raised against one ormore immunogenic polypeptides of the present invention. Unless otherwisestated, it is to be understood that the antibody of the presentinvention may include a mixture of two or more different types ofantibody. For example, the antibody may be a mixture of two types ofantibodies, wherein one of the two types specifically binds to oneparticular antigen and the other of the two types specifically binds tosome other antigen.

The “immunogenic polypeptide of the present invention” and, more simply,“the polypeptide of the present invention”, is an immunogen againstwhich the antibodies of the present invention may be raised. All“polypeptides of the present invention” are immunogenic and thereforemay be used to elicit an immune response in a host animal to produce theantibodies of the present invention. Unless otherwise stated, it is tobe understood that the polypeptide of the present invention may be onecomponent of a mixed composition of a plurality of components.

An “immunogen” is any agent, such as the immunogenic polypeptide of thepresent invention, for example, that is capable of eliciting an immuneresponse in an animal that is exposed to that agent.

The term “roundworm”, as used herein, refers to helminths such asintestinal roundworms of the order Ascaridida, which includes the generaToxocara, Toxascaris, Baylisascaris, Ascaridia, Parascaris, Ascaris,Anisakis, and Pseudoterranova. Thus, the term “roundworm”, as usedherein, does not refer to the entirety of the phylum Nematoda.Therefore, “roundworm” does not include any member of the generaAncylostoma, Uncinaria, Necator, Trichuris or Dirofilaria.

A “roundworm coproantigen” or a “coproantigen of roundworm” is anyroundworm product that is present in the feces of a mammal having aroundworm infection and that may be specifically bound by one or more ofthe antibodies of the invention. For example, a roundworm coproantigenmay be, but is not limited to being, one or more of the polypeptides ofthe invention.

“Specific for”, “specifically binds”, and “stably binds” means that aparticular composition of the invention, such as an antibody,polypeptide, or oligonucleotide of the present invention, for example,recognizes and binds to one or more other agents with greater affinitythan to at least one other agent. As one example, an antibody of thepresent invention is said to be “specific for”, to “specifically bind”,and to “stably bind” roundworm antigens whenever that antibody is ableto recognize and bind to those roundworm antigens with greater affinitythan to any other antigens from a non-roundworm parasitic worm. Suchbinding specificity can be tested using methodology well known in theart, for example, ELISA or a radioimmunoassay (RIA). Based oninformation observed regarding the binding specificity of a particularcomposition of the invention, the method of the present invention can becarried out under conditions that allow that composition to bind to (andtherefore to allow the detection of such binding to) a particular agentor agents, but not to significantly bind other agents, while thoseconditions are maintained. As one example, the method of the presentinvention can be carried out under conditions that allow an antibody ofthe present invention to bind to (and therefore to allow the detectionof such binding to) one or more roundworm antigens present in aparticular sample, but not significantly to any hookworm, whipworm orheartworm antigen that may be present in that sample.

“Detecting roundworm” means detecting one or more roundworm-specificproducts, including one or more of the polypeptides, antibodies andnucleic acids of the present invention, or one or more roundwormantigens, for example. The presence of one or more such roundwormproducts in a sample from a mammal is indicative that the mammal has aroundworm infection, regardless of whether any whole roundworm organismor ovum thereof is also present in that sample. Conversely, the absenceof one or more such roundworm products a sample from a mammal isindicative that the mammal does not have a roundworm infection.

“Amino acid” refers to naturally occurring and synthetic amino acids.Amino acid residues are abbreviated as follows: Alanine is A or Ala;Arginine is R or Arg; Asparagine is N or Asn; Aspartic Acid is D or Asp;Cysteine is C or Cys; Glutamic Acid is E or Glu; Glutamine is Q or Gln;Glycine is G or Gly; Histidine is H or H is; Isoleucine is I or Ile;Leucine is L or Leu; Lysine is K or Lys; Methionine is M or Met;Phenylalanine is F or Phe; Proline is P or Pro; Serine is S or Ser;Threonine is T or Thr; Tryptophan is W or Trp; Tyrosine is Y or Tyr; andValine is V or Val. Except where defined otherwise herein, X or Xaarepresents any amino acid. Other relevant amino acids include, but arenot limited to being, 4-hydroxyproline and 5-hydroxylysine. In allcases, the amino acid sequence of a polypeptide described or otherwisereferred to herein is presented in conventional form in that theleft-most, or first, amino acid residue of the sequence is theN-terminal residue and the right-most, or last, amino acid residue ofthe sequence is the C-terminal residue.

A “conservative variant” of any particular nucleic acid sequenceincludes any sequence having one or more degenerate codon substitutionsto that particular nucleic acid sequence, any sequence having one ormore nucleotide substitutions to, insertions to, and deletions from thatparticular nucleic acid sequence, and the complementary sequence of thatparticular nucleic acid and the conservative variants of thatcomplementary sequence. Conservative variants of a particular nucleicacid sequence preferably have at least about 85% identity, morepreferably have at least about 90% identity, and even more preferably atleast about 95-99% identity, to that particular nucleic acid sequence.Conservative variants of a particular nucleic acid sequence may beartificially synthesized or they may be isolated in their natural formfrom an organism, including from a roundworm organism, such as Toxocaracanis and Toxocara cati, for example.

A “conservative variant” of any particular polypeptide sequence is anypolypeptide having an amino acid sequence that varies from the aminoacid sequence of that particular polypeptide but still retains thespecific binding properties of that particular polypeptide, such that anantibody of the present invention that is raised against the particularpolypeptide is capable of specifically binding the variant polypeptide.Therefore, for example, a conservative variant of a particularpolypeptide may have one or more amino acid substitutions, deletions,additions, and insertions to that particular polypeptide. For example, aconserved variant of a particular polypeptide may have 30 or fewer, 25or fewer, 20 or fewer, 15 or fewer, 10 or fewer, or 5 or fewer,conserved amino acid substitutions to that particular polypeptide.Conservative variants of a particular polypeptide preferably, but notessentially, have at least about 80% identity, more preferably have atleast about 90% identity, and even more preferably at least about 91-99%identity, to that particular polypeptide. A percent identity for anysubject nucleic acid or amino acid sequence (e.g., any of polypeptidesdescribed herein) relative to another “target” nucleic acid or aminoacid sequence can be determined as follows. First, a target nucleic acidor amino acid sequence of the invention can be compared and aligned to asubject nucleic acid or amino acid sequence, using the BLAST 2 Sequences(B12seq) program from the stand-alone version of BLASTZ containingBLASTN and BLASTP (e.g., version 2.0.14). The stand-alone version ofBLASTZ can be obtained at www.ncbi.nlm.nih.gov. Instructions explaininghow to use BLASTZ, and specifically the B12seq program, can be found inthe ‘readme’ file accompanying BLASTZ. The programs also are describedin detail by Karlin et al. (1990) Proc. Natl. Acad. Sci. 87:2264; Karlinet al. (1990) Proc. Natl. Acad. Sci. 90:5873; and Altschul et al. (1997)Nucl. Acids Res. 25:3389.

B12seq performs a comparison between the subject sequence and a targetsequence using either the BLASTN (used to compare nucleic acidsequences) or BLASTP (used to compare amino acid sequences) algorithm.Typically, the default parameters of a BLOSUM62 scoring matrix, gapexistence cost of 11 and extension cost of 1, a word size of 3, anexpect value of 10, a per residue cost of 1 and a lambda ratio of 0.85are used when performing amino acid sequence alignments. The output filecontains aligned regions, of homology between the target sequence andthe subject sequence. Once aligned, a length is determined by countingthe number of consecutive nucleotides or amino acid residues (i.e.,excluding gaps) from the target sequence that align with sequence fromthe subject sequence starting with any matched position and ending withany other matched position. A matched position is any position where anidentical nucleotide or amino acid residue is present in both the targetand subject sequence. Gaps of one or more residues can be inserted intoa target or subject sequence to maximize sequence alignments betweenstructurally conserved domains (e.g., α-helices, β-sheets, and loops).

The percent identity over a particular length is determined by countingthe number of matched positions over that particular length, dividingthat number by the length and multiplying the resulting value by 100.For example, if (i) a 500 amino acid target sequence is compared to asubject amino acid sequence, (ii) the B12seq program presents 200 aminoacids from the target sequence aligned with a region of the subjectsequence where the first and last amino acids of that 200 amino acidregion are matches, and (iii) the number of matches over those 200aligned amino acids is 180, then the 500 amino acid target sequencecontains a length of 200 and a sequence identity over that length of 90%(i.e., 180/200×100=90). It will be appreciated that a nucleic acid oramino acid target sequence that aligns with a subject sequence canresult in many different lengths with each length having its own percentidentity. It is noted that the percent identity value can be rounded tothe nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 isrounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 isrounded up to 78.2. It is also noted that the length value will alwaysbe an integer.

Conservative variants of a particular polypeptide sequence may beartificially synthesized or they may be isolated in their natural formfrom an organism, including from a roundworm organism, such as Toxocaracanis and Toxocara cati, for example. In one specific example, thepolypeptide of the invention having an amino acid sequence correspondingto SEQ ID NO:4 shown below is a conservative variant of the polypeptideof the present invention having an amino acid sequence corresponding toSEQ ID NO:3 in that SEQ ID NO:4 is more than 92% identical to SEQ IDNO:3 over an alignment of 131 amino acids. More generally, each one ofSEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 areconserved variants of each other. It is also to be understood that otherconserved variants of the SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6 and SEQ ID NO:7 are contemplated by the present invention asdescribed herein, but the skilled artisan would recognize that all ofthese contemplated variants are too numerous to list. The skilledartisan will also recognize that these variants include, but are notlimited to, those have one or more substitutions of basic amino acidresidues, one or more substitutions of acidic amino acid residues, oneor more substitutions of polar amino acid residues, one or moresubstitutions of hydrophobic amino acid residues, one or moresubstitutions of aromatic amino acid residues, and one or moresubstitutions of small amino acid residues. (“Basic” amino acid residuesare K, R and H. “Acidic” amino acid residues are D and E. “Polar” aminoacid residues are N and Q. “Hydrophobic” amino acids are I, L, and V.“Aromatic” amino acid residues are F, Y, and W. “Small” amino acids areG, S, A, T and M.)

III. Nucleic Acids and Polypeptides of the Invention

The nucleic acids and polypeptides of the invention are described indetail in Provisional Application: “Methods, Devices, Kits AndCompositions For Detecting Roundworm,” Application Ser. No. 61/128,079,filed May 19, 2008, and is incorporated by reference in its entirety.

In an attempt to identify compositions that may be used to confirm thepresence or absence of roundworm in a mammalian sample, a plurality ofoligonucleotide primers corresponding to portions of two expressedsequence tag (EST) clones (ko15fo7 and ko34f08; which are available fromthe Washington University Genome Sequencing Center, St. Louis, Mo.) weredesigned, synthesized and used in 5′ RACE, 3′RACE and RT-PCR reactionsthat included total RNA isolated from either Toxocara canis or Toxocaracati. As a result of these efforts, an 865-nucleotide cDNA sequence wasdeduced from Toxocara canis (this sequence is shown in FIG. 1 and isidentified herein as SEQ ID NO: 1), and a 632-nucleotide cDNA sequencewas deduced from Toxocara cati (this sequence is shown in FIG. 2 and isidentified herein as SEQ ID NO:2). (BLAST. searches that were carriedout using SEQ ID NO: 1 indicated that a portion of that sequence issimilar to, but is not identical to or substantially identical to,nucleic acid sequence that is predicted to encode a portion of the TBA-1protein of Toxocara canis and to nucleic acid sequence that is predictedto encode the ABA-1 protein of Ascaris lumbricoides (see S. Yahiro etal., Parasite Immunology 20:351-7 (1998); M. W. Kennedy, ParasitologyToday 16:373-80 (2000); and Y. Xia et al., Parasitology 120:211-24(2000).)

Analysis of the sequences corresponding to SEQ ID NO: 1 and SEQ ID NO:2indicated that each one of these sequences contains a large ORF.Specifically, as shown in FIG. 3, the large ORF of SEQ ID NO: 1corresponds to nucleotides 2 through 616 of SEQ ID NO: 1 and ispredicted to encode a polypeptide having the following amino acidsequence:

(SEQ ID NO: 3) KKIYGVAASRRRRHHFTLENSLDTHLKWLSHEQKEELLQMKKDGKSKKELQDKIMHYYEHLEGDAKHEATEQLKGGCREILKHVVGEEKAAEIKALKDSGASKDELKAKVEEALHAVTDEEKKQHIAEFGPACKKIYGVAASRRRRHHFTLENSLDTHLKWLSHEQKEELLQMKKDGKSKKELQDKIMHYYEHLEGMLLA LCILY.

Further, as shown in FIG. 4, the large ORF of SEQ ID NO:2 corresponds tonucleotides 1 through 486 of SEQ ID NO:2 and is predicted to encode apolypeptide having the following amino acid sequence:

(SEQ ID NO: 4) IYGVAASRRRRHHFTLEKSLDTHLKWLSHEQKEELLKMKKDGKSKKELQDKVMHFYEHLEGDAKHEATEQLKGGCREILKHVVGEEKAAEIKALKDSGASKDELKAKVEDALHAVTDEEKKQHIAEFGPACKEIFGVPIDVRHKRDPYTN MTPDEVAEGLRS.

The polypeptides of the present invention may be encoded for by nucleicacids that have a nucleotide sequence that corresponds to all orportions of SEQ ID NO: 1 and SEQ ID NO:2 or to all or portions of anyconservative variant of those sequences. It is to be understoodtherefore that the amino acid sequence of the polypeptide of the presentinvention is variable.

For example, the polypeptide of the present invention may have an aminoacid sequence that corresponds to all or a portion of SEQ ID NO:3 or SEQID NO:4 or all or a portion of any conservative variant of SEQ ID NO:3or SEQ ID NO:4.

In one specific example, the polypeptide of the present invention hasthe following amino acid sequence:

(SEQ ID NO: 5) MHHFTLENSLDTHLKWLSHEQKEELLQMKKDGKSKKELQDKIMHYYEHLEGDAKHEATEQLKGGCREILKHVVGEEKAAEIKALKDSGASKDELKAKVEEALHAVTDEEKKQHIAEFGPACKKIYGVAAS.

The 129 amino acid residues that follow the N-terminal methionineresidue of the polypeptide corresponding to SEQ ID NO:5 specificallyrepresent the amino acid residues 14 through 142 of SEQ ID NO:3. Asdescribed in the Example section included herein, the N-terminalmethionine was artificially added to the N-terminus of this polypeptideby carrying out a standard cloning technique. Also as describedthroughout the Example section, antibody raised against the polypeptidecorresponding to SEQ ID NO:5 was useful for detecting roundworm antigen.Because the N-terminal methionine was artificially added, and is notthought to naturally exist in Toxocara (the residue that is immediatelyprior to the histidine residue at position 14 in each one of SEQ ID NO:3and SEQ ID NO:4 is arginine, and not methionine), it is thereforecontemplated that the polypeptide of the present invention may have anamino acid sequence that corresponds to amino acid residues 14 through142 of SEQ ID NO:3, or, more specifically:

(SEQ ID NO: 6) HHFTLENSLDTHLKWLSHEQKEELLQMKKDGKSKKELQDKIMHYYEHLEGDAKHEATEQLKGGCREILKHVVGEEKAAEIKALKDSGASKDELKAKVEEALHAVTDEEKKQHIAEFGPACKKIYGVAAS.

Further, an alignment of SEQ ID NO:5 (mostly Toxocara canis-derivedsequence; with the only exception being the N-terminal methionineresidue) to SEQ ID NO:4 (Toxocara cati-derived sequence) is shown inFIG. 5. Because antibody raised against a polypeptide having sequencecorresponding to SEQ ID NO:5 was useful for detecting Toxocara cati (seethe Example section included herein), it is additionally contemplatedthat the polypeptide of the present invention may have the amino acidsequence corresponding to SEQ ID NO:7 (which also is shown in FIG. 5),wherein the X at position 1 is I or absent, the X at position 2 is Y orabsent, the X at position 3 is G or absent, the X at position 4 is V orabsent, the X at position 5 is A or absent, the X at position 6 is A orabsent, the X at position 7 is S or absent, the X at position 8 is R orabsent, the X at position 9 is R or absent, the X at position 10 is R orabsent, the X at position 11 is R or M, the X at position 18 is N or K,the X at position 37 is Q or K, the X at position 52 is I or V, X atposition 55 is Y or F, the X at position 110 is E or D, the X atposition 133 is K or E, the X at position 135 is Y or F, the X atposition 138 is A or P, the X at position 139 is A or I, the X atposition 140 is S or D, the X at position 141 is V or absent, the X atposition 142 is R or absent, the X at position 143 is H or absent, the Xat position 144 is K or absent, the X at position 145 is R or absent,the X at position 146 is D or absent, the X at position 147 is P orabsent, the X at position 148 is Y or absent, the X at position 149 is Tor absent, the X at position 150 is N or absent, the X at position 151is M or absent, the X at position 152 is T or absent, the X at position153 is P or absent, the X at position 154 is D or absent, the X atposition 155 is E or absent, the X at position 156 is V or absent, the Xat position 157 is A or absent, the X at position 158 is E or absent,the X at position 159 is G or absent, the X at position 160 is L orabsent, the X at position 161 is R or absent, and the X at position 162is S or absent.

It is also contemplated that any one or more of the SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 may be only a portion ofa larger polypeptide sequence, and therefore may represent partialsequence of one or more proteins that normally are expressed inroundworm, for example, or one or more polypeptide sequences that areartificially fused to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6and SEQ ID NO:7. The skilled artisan will recognize that are a varietyof techniques exist for artificially fusing two or more polypeptidefragments together.

It is even further contemplated that the polypeptide of the presentinvention may include more than one of the SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6 and SEQ ID NO:7. For example, the polypeptide ofthe present invention may include the SEQ ID NO:5 fused to the SEQ IDNO:7. Also, it is contemplated that the polypeptide of the presentinvention may include a plurality of polypeptide fragments correspondingto SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.For example, the polypeptide of the present invention may be formed by aplurality of polypeptide fragments corresponding to SEQ ID NO:5 that arefused together. In another example, the polypeptide of the presentinvention may be formed by a plurality of polypeptide fragmentscorresponding to SEQ ID NO:5 and a plurality of polypeptide fragmentscorresponding to SEQ ID NO:7 that are fused together in any combination.

Whereas one particular polypeptide of the present invention wasexpressed and isolated by a specific technique (in which is described inthe Example section included herein), the skilled artisan will recognizethat any of the polypeptides of the present invention may be isolated byemploying any one or more of a variety of techniques. (See, e.g., Sewaldand Jakubke, Peptides: Chemistry and Biology, Wiley Publishing (2002);Peptide Synthesis and Applications (Methods in Molecular Biology) Howl,ed., Humana Press (2005); Jones, Amino Acid and Peptide Synthesis,Oxford University Press (2002), each one of which is incorporated hereinby reference in its entirety.) These techniques include those that maybe carried out to isolate naturally existing polypeptides having aminoacid sequence corresponding to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6 and SEQ ID NO:7 and any naturally occurring variant of thosepolypeptides. These techniques further include those that may be carriedout to artificially generate the polypeptides having amino acid sequencecorresponding to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 andSEQ ID NO:7 and any conserved variant of those polypeptides. Suchvariants may be generated, for example, by employing any one or moremutagenesis techniques or by direct synthesis.

The polypeptides of the present invention are capable of eliciting animmune response in a host animal that is exposed to these polypeptidesto produce one or more of the antibodies of the present invention.Regardless of the technique by which they are derived, the polypeptidesof the present invention are preferably prepared in substantially pureform when they are to be used for the purpose of raising antibody.Preferably, these polypeptides are at least about 80% pure, morepreferably are at least about 90-95% pure, and even more preferably areat least about 99% pure. Exemplary techniques for eliciting an immuneresponse in a host organism and for isolating antibodies therefrom aredescribed herein, but it is to be understood that the present inventionis not limited to those techniques. The skilled artisan will recognizethat there are a plurality of techniques for achieving this same goalwithout deviating from the scope and spirit of the invention.

IV. Antibodies of the Invention

The present invention further includes antibodies and antigen-bindingfragments thereof that are raised against and that specifically bind allor part of one or more polypeptides of the present invention, and alsoincludes compositions that include said antibodies and antigen-bindingfragments thereof. When contacted to a sample obtained from a mammal,these antibodies and antigen-binding fragments are able to specificallybind roundworm antigen present in the sample, but are not able tospecifically bind any antigen from hookworm, whipworm, or heartworm thatmay be present in the sample. The antibodies of the present inventionare suitable for being used only to capture one or more roundwormantigens, only to detect one or more roundworm antigens, or morepreferably, to both capture and detect one or more roundworm antigens.

The antibodies of the present invention may belong to any antibodyclass, including for example, IgG, IgM, IgA, IgD and IgE, and may beprepared by any of a variety of techniques known to the skilled artisan.(See, e.g., Dean, Methods Mol. Biol. 80:23-37 (1998); Dean, Methods Mol.Biol. 32:361-79 (1994); Baileg, Methods Mol. Biol. 32:381-88 (1994);Gullick, Methods Mol. Biol. 32:389-99 (1994); Drenckhahn et al. MethodsCell. Biol. 37:7-56 (1993); Morrison, Ann. Rev. Immunol. 10:239-65(1992); Wright et al. Crit. Rev. Immunol. 12:125-68 (1992); Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory(1988); and Making and Using Antibodies: A Practical Handbook, Howardand Kaser, eds., CRC Press (2006), each one of which is incorporatedherein by reference in its entirety.)

In one technique, the polypeptide of the invention is introduced into ahost animal, such as into rabbit, mouse, rat, guinea pig, goat, pig,cow, sheep, donkey, dog, cat, chicken, or horse, for example. Anenhanced immune response may be elicited in the host animal byassociating the polypeptide with a carrier and/or by exposing the hostto an adjuvant, but it is to be understood that the present inventiondoes not require that the polypeptide be associated with a carrier orthat the host be exposed to the adjuvant. An exemplary carrier that maybe used for this purpose is bovine serum albumin, bovine thyroglobulin,and soybean trypsin inhibitor. Exemplary adjuvants include Freund'scomplete or incomplete adjuvant and MDL-TDM adjuvant. Regardless ofwhether the polypeptide is associated with such a carrier or whether thehost is exposed to an adjuvant, booster immunizations optionally may bemade with the host animal being bled one or more times thereafter.Polyclonal antibodies that specifically bind the polypeptide may then bepurified from antisera obtained from the bleed or bleeds. Suchpurification may be achieved, for example, by employing affinitychromatography techniques that involve associating the polypeptide to asolid support. Such affinity chromatography techniques are well known bythe skilled artisan.

In one embodiment, the antibody of the present invention is an antibodythat is raised in rabbit by immunizing that host animal with thepolypeptide having the amino acid sequence corresponding to SEQ ID NO:5.(Hereinafter, this particular antibody is referred to as“anti-DIV6716”.) A specific technique for producing and isolatinganti-DIV6716 pAB is described in the Example section included herein,but the skilled artisan will recognize that the production and isolatingof anti-DIV6716 pAB, or any other antibody of the present invention, isnot limited to that specific technique.

In other embodiments, the antibody of the present invention is raised ina host against one or more polypeptides having an amino acid sequencethat is a conservative variant of the sequence corresponding to SEQ IDNO:5. In some other embodiments, the antibody of the present inventionis raised in a host against any one or more polypeptides having an aminoacid sequence corresponding to the sequence of SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:6, or SEQ ID NO:7, or one or more polypeptides having an aminoacid sequence that is a conservative variant of any of those sequences.

In another embodiment, the antibody of the present invention is anantibody that specifically binds one or more the polypeptide having theamino acid sequence corresponding to SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, or SEQ ID NO:7, or antigenic portions thereof.

In yet other embodiments, the antibody of the present inventionspecifically binds one or more polypeptides having an amino acidsequence that is a conservative variant of the sequence corresponding toSEQ ID NO:5. In some other embodiments, the antibody of the presentinvention specifically binds one or more polypeptides having an aminoacid sequence corresponding to the sequence of SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:6, or SEQ ID NO:7, or one or more polypeptides having an aminoacid sequence that is a conservative variant of any of those sequences.

It is also to be understood that the antibodies of the invention may bepolyclonal or monoclonal antibodies, single chain antibodies (scFv),chimeric antibodies, and fragments thereof. Monoclonal antibodies thatare specific for the polypeptide of interest may be obtained andpurified, for example, by preparing cell lines that generate antibodieshaving the desired specificity to the polypeptide of interest. Celllines of this kind may be derived from cells of a particular type (e.g.,spleen cells) that are isolated from a host animal that had previouslybeen immunized with the polypeptide as described before. In such a case,these cells could then be immortalized, for example, by fusing them withmyeloma cells by carrying out any one of a variety of fusion techniquesknown to the skilled artisan. In one exemplary technique, the cells fromthe immunized host animal are co-incubated with their fusion partner,e.g., the myeloma cells, in the presence of a detergent for a shortperiod of time before being plated on a medium that supports the growthof hybrid cells (but not the myeloma fusion partner). Such selection maybe achieved, for example, by using hypoxanthine, aminopterin, andthymidine (HAT). When hybrid cells emerge during selection, in perhapsone or two weeks after commencing the selection process, single hybridcolonies (and their supernatants) are tested for their ability to bindthe polypeptide or polypeptides against which the host animal wasimmunized. Hybrid colonies having the most optimal binding specificitywould represent the best candidates from which monoclonal antibodies maybe isolated. These monoclonal antibodies, for example, may be isolateddirectly from the supernatant (i.e., medium) in which these colonies aregrown by employing any one of a variety techniques known to the skilledartisan.

The antibodies of the invention also may be a single chain antibody(scFv), or an antigen binding fragment of an antibody. Antigen-bindingfragments of antibodies are a portion of an intact antibody comprisingthe antigen binding site or variable region of an intact antibody,wherein the portion is free of the constant heavy chain domains of theFc region of the intact antibody. Examples of antibody fragments includeFab, Fab′, Fab′-SH, F(ab′)₂ and F_(v) fragments. In addition toproduction and purification from animals or mammalian cells, antibodies,antibody fragments, or non-antibody scaffolds can be selected based uponvarious in vitro technologies, including phage display, ribosomaldisplay, or bacterial display.

Antibodies, including secondary antibodies, may be labeled with any typeof label known in the art, including, for example, fluorescent,chemiluminescent, radioactive, enzymes, colloidal particles,radioisotopes and bioluminescent labels. In various embodiments of theinvention, the one or more of the antibodies of the invention arelabeled with an enzyme, a colloidal particle, a radionuclide or afluorophor. The particulate label can be, for example, a colored latexparticle, dye sol, or gold sol conjugated to an antibody.

V. Methods, Devices and Kits of the Invention

A. Devices and Kits of the Invention

The present invention, in one aspect, is a device for the detection ofroundworm infection in a mammal, such as a canine, feline, porcine,bovine, or human, for example. The device is arranged to aid in thedetection of the presence or absence of roundworm antigen in a samplefrom a mammal that may also be infected with one or more other wormparasites, including hookworm, whipworm, and heartworm.

In one aspect, the device includes a solid support, wherein one or moreantibodies of the invention are immobilized on the solid support. Thesolid support may be, but is not limited to being, the inner, bottomsurface of a well of a microtiter plate or a substrate that is includedas part of a lateral flow device, for example. An exemplary microtiterplate is an Immulon 1B 96-well plate (which is commercially availablefrom Thermo Scientific of Milford, Mass.), but it is to be understoodthat the skilled artisan will recognize that a large variety of othermicrotiter plates that are not the Immulon 1B 96-well plate allow forthe immobilization of antibodies thereon, and therefore would besuitable for providing the solid support of the present invention.

An exemplary lateral flow device is the lateral flow device that isdescribed in U.S. Pat. No. 5,726,010, which is incorporated herein byreference in its entirety. The device for performing a lateral flowassay may be a SNAP® device, which is commercially available from IDEXXLaboratories, Inc. of Westbrook, Me. However, it is to be understoodthat the skilled artisan will recognize that a large variety of otherlateral flow devices that are not SNAP® devices or described by U.S.Pat. No. 5,726,010 allow for the immobilization of an antibody thereon,and therefore would be suitable for being used as the device of thepresent invention. These devices can include, for example, lateral flowdevices that use colloidal gold technology.

Antibodies used in the device of the invention may be immobilized on thesolid support by any methodology known in the art, including, forexample, covalently or non-covalently, directly or indirectly, attachingthe antibodies to the solid support. Therefore, while these antibodiesmay be attached to the solid support by physical adsorption (i.e.,without the use of chemical linkers), it is also true that theseantibodies may be immobilized to the solid support by any chemicalbinding (i.e., with the use of chemical linkers) method readily known toone of skill in the art.

It is also to be understood that the solid support may be any suitablematerial for the immobilization of the antibodies of the invention. Forexample, the solid support may be beads, particles, tubes, wells,probes, dipsticks, pipette tips, slides, fibers, membranes, papers,natural and modified celluloses, polyacrylamides, agaroses, glass,polypropylene, polyethylene, polystyrene, dextran, nylon, amylases,plastics, magnetite or any other suitable material readily known to oneof skill in the art.

The device optionally may include one or more labeled antigen capturereagents that may be mixed with a sample from a mammal prior toapplication to a device of the invention. When the labeled captureantigen reagent is included, the labeled antigen capture reagent may ormay not be deposited or dried on a solid surface of the device. “Antigencapture reagent” refers to any compound that is specific for the antigenor antigens of interest. The labeled antigen capture reagent, whetheradded to the mammalian sample or pre-deposited on the device, may be,for example, a labeled antibody specific for a roundworm antigen,including, but not limited to, the antibodies of the present invention.In just one example, anti-DIV6716 pAB conjugated with horseradishperoxidase may be used as a labeled antigen capture reagent.

The device also may optionally include a liquid reagent that transports(such as when the device is a SNAP® device, for example), or otherwisefacilitates removal of (such as when the device includes a microtiterplate, for example), unbound material (e.g., unreacted portions of themammalian sample, such as, for example, unreacted portions of fecalextract, and unbound antigen capture reagent) away from the reactionzone (solid phase). The liquid reagent may be a wash reagent and serveonly to remove unbound material from the reaction zone, or it mayinclude a detector reagent and serve to both remove unbound material andfacilitate antigen detection. For example, in the case of an antigencapture reagent conjugated to an enzyme, the detector reagent includes asubstrate that produces a detectable signal upon reaction with theenzyme-antibody conjugate at the reaction zone (solid phase).Alternatively, in the case of a labeled antigen capture reagentconjugated to a radioactive, fluorescent, or light-absorbing molecule,the liquid reagent acts merely as a wash solution facilitating detectionof complex formation at the reactive zone by washing away unboundlabeled reagent.

The liquid reagent may further include a limited quantity of an“inhibitor”, i.e., a substance that blocks the development of thedetectable end product. A limited quantity is defined as being an amountof inhibitor sufficient to block end product development until most orall excess, unbound material is transported away from the second region,at which time detectable end product is produced.

The device of the present invention may also include various bindingreagents immobilized at locations distinct from the antigen capturereagent or reagents. For example, an immunoreagent (an antibody, antigenor polypeptide) that recognizes a species-specific (e.g.,roundworm-specific) antibody portion of a labeled antibody or antigencapture reagent, or an enzyme portion of an enzyme-labeled reagent, canbe included as a positive control to assess the viability of thereagents within the device. For example, a positive control may be ananti-horseradish peroxidase antibody that has been raised in, forexample, goat or mouse. Additionally, a reagent, e.g., an antibody,isolated from a non-immune member of the species from which the antibodyportion of the antigen-antibody complex was derived can be included as anegative control to assess the specificity of immunocomplex (i.e.,antigen-antibody complex) formation.

In addition to being designed to detect roundworm in a mammalian sample,the device of the invention optionally may be designed to allow one ormore other diagnostic tests to be performed. For example, the solidsupport may also include reagents for the detection of one or morenon-roundworm worm parasites, one or more non-worm parasites, one ormore viruses, one or more fungi, or one or more bacteria. The reagentsfor the detection of one or more non-roundworm worm parasites, one ormore non-worm parasites, one or more viruses, one or more fungi, or oneor more bacteria may be, for example, one or more antibodies or one ormore antigens recognized by antibodies specific for one or morenon-roundworm worm parasites, one or more non-worm parasites, one ormore viruses, one or more fungi, or one or more bacteria.

In one embodiment, which is shown in FIGS. 6A and 6B, the device of thepresent invention is a microtiter plate 10 that includes a plurality ofwells 12, wherein each well 12 includes a solid support 14 havinganti-DIV6716 pAB (represented as element 16) immobilized thereupon.

The plate 10 may be used in conjunction with a method of the presentinvention to detect roundworm in a mammalian sample. Specifically, aroundworm infection may be diagnosed in a mammal by detecting one ormore roundworm antigens with the anti-DIV6716 pAB that is immobilized onthe solid support 14. In one embodiment, the antigens that are detectedare roundworm coproantigens. “Roundworm coproantigens” are any productor products of roundworm that are present in a fecal sample and that canspecifically and stably bind to the anti-DIV6716 pAB. Roundwormcoproantigens therefore may be whole roundworm, roundworm eggs,roundworm fragments, or products secreted, excreted or shed fromroundworm or a combination thereof. Roundworm coproantigens furtherinclude the polypeptides of the present invention, such as thepolypeptides having an amino acid sequence corresponding to SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, polypeptideshaving an amino acid sequence that is a conservative variant of thosesequences, and/or antigenic fragments of any such polypeptides, forexample.

The invention further includes assay kits (e.g., articles ofmanufacture) for detecting roundworm in a mammalian sample. A kittherefore may include one or more devices and/or compositions of thepresent invention. For example, the kit may include anti-roundwormantibodies and means for determining binding of the antibodies toroundworm antigens in the sample. In one particular example, such a kitincludes the device having an immobilized anti-roundworm antibody, suchas anti-DIV6716 pAB, for example, one or more antigen capture reagents(e.g., a non-immobilized labeled antigen capture reagent and animmobilized antigen capture reagent) and wash reagent, as well asdetector reagent and positive and negative control reagents, if desiredor appropriate. Other components such as buffers, controls, and thelike, known to those of ordinary skill in art, may be included in suchtest kits. The relative amounts of the various reagents can be varied,to provide for concentrations in solution of the reagents thatsubstantially optimize the sensitivity of the assay. Particularly, thereagents can be provided as dry powders, usually lyophilized, which ondissolution will provide for a reagent solution having the appropriateconcentrations for combining with a sample. The present kit may furtherinclude instructions for carrying out one or more methods of the presentinvention, including instructions for using any device and/orcomposition of the present invention that is included with the kit.

B. Methods of the Invention

The present invention further includes methods for using one or more ofthe devices, kits and/or compositions of the present invention to detectthe presence or absence of roundworm in a sample. The methods thereforemay be carried out to detect the presence or absence of roundworm in asample, such as, for example, a fecal sample, that is obtained from amammal, including, but not limited to, a canine, feline, porcine, bovineor human. Further, the methods may be carried out to detect Toxocara,such as T. canis or T. cati, or T. vitulorum, for example. It is to beunderstood, however, that these methods are not limited to being used todetect Toxocara, and therefore these methods may be carried out for thepurpose of detecting other species of roundworm, such as Toxascaris,including T. leonina, Baylisascaris, including B. procyonis, Ascaridia,including A. galli, Parascaris, including P. equorum, Ascaris, includingA. lumbricoides and A. suum, Anisakis, including Anisakis simplex, orPseudoterranova, including P. decipiens, for example. These methodsfurther are useful for confirming such presence or absence of roundwormin a sample even when that sample includes one or more products derivedfrom other worm species, including one or more products from hookworm,whipworm, and/or heartworm.

In the methods of the present invention, detection of roundworm may beaccomplished by detecting the presence or absence of one or moreroundworm antigens, such as the polypeptides having an amino acidsequence corresponding to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, or SEQ ID NO:7, as well as antigenic fragments and/or conservativevariants of those sequences, for example. When the sample under test forroundworm is feces, the soluble portion of the feces may be collected byany protocol known in art. For example, in addition to the specificprotocol described in the Example section herein, the soluble portionsof the sample generally may be collected by using filtration,extraction, centrifugation, or simple mixing followed by gravimetricsettling. The skilled artisan will recognize that there are a variety ofways of extracting and preparing non-fecal samples from a mammal aswell. For example, the sample may be a bodily fluid that is naturallyexcreted or otherwise released by the mammal or that is artificiallyobtained from the mammal. Such artificial extraction may be carried outby milking the mammal or by injecting a syringe into the mammal anddrawing the fluid into the syringe. Once obtained, the fluid optionallymay be fractionated (for example, serum may be fractionated from wholeblood as then used as the sample). As another example, the sample may beobtained by swabbing the mammal, such as the oral cavity of the mammal,for example. As yet another example, tissue sections may be obtained bybiopsy.

The methods include contacting the mammalian sample with one or moreantibodies specific for one or more roundworm antigens under conditionsthat allow an antigen/antibody complex, i.e., an immunocomplex, to form.That is, an antibody specifically binds to a roundworm antigen presentin the sample. The skilled artisan is familiar with assays andconditions that may be used to detect such antigen/antibody complexbinding. For example, the antigen/antibody complex may be detected usinga secondary antibody that binds to the antigen/antibody complex. Theformation of a complex between roundworm antigen and anti-roundwormantibodies in the sample may be detected using any suitable method knownin the art.

Further, the relative amount of antibody-antigen complexes that areformed in one particular reaction may be measured with respect to thoseformed in any other reaction by any methodology known in the art forachieving that goal. When it is determined that a sample under test hasmore antibody-antigen complexes than does a control sample, it can beconcluded that roundworm is present in the test sample. When this istrue, it may be concluded that the mammal from which the test sample wasobtained harbors an intestinal roundworm infection. Either one or bothof the conclusions that roundworm is present in the test sample and thatthe mammal being tested harbors an intestinal roundworm infection may bemade by a clinician at a diagnostic service provider or by a caregiverof the mammal, such as the mammal's veterinarian, for example. When acaregiver of a mammal determines (or is otherwise informed that) amammal harbors a roundworm infection, the caregiver may then subject themammal to a course of treatment that is optimally designed to rid themammal of roundworm specifically, rather than of a parasitic nematodeinfection generally. In addition, humans who may come in contact withthe infested animal or its excretions may be advised to take precautionsagainst acquiring the parasite. In this context, it is important todetermine the worm species with high specificity, as some helminths,such as roundworms and hookworms, can cause significant disease (e.g.,larval migrans) in humans, while it is generally accepted that whipwormdoes not play a zoonotic role of importance in humans. Further, thepresent invention can be used to confirm that any animal that hasreceived treatment for a roundworm infection has been rid of thatinfection.

The steps of the method of the present invention may include applying amammalian sample to a device of the invention, which includes animmobilized antibody specific for one or more roundworm antigens, anddetecting the presence or absence of the roundworm antigen in thesample. Antibodies specific for antigens of roundworms may be directlyor indirectly attached to a solid support or a substrate such as amicrotiter well, antibody-immobilizing portion of a SNAP® device,magnetic bead, non-magnetic bead, column, matrix, membrane, fibrous matcomposed of synthetic or natural fibers (e.g., glass or cellulose-basedmaterials or thermoplastic polymers, such as, polyethylene,polypropylene, or polyester), sintered structure composed of particulatematerials (e.g., glass or various thermoplastic polymers), or castmembrane film composed of nitrocellulose, nylon, polysulfone or the like(generally synthetic in nature). All of these substrate materials may beused in suitable shapes, such as films, sheets, or plates, or they maybe coated onto or bonded or laminated to appropriate inert carriers,such as paper, glass, plastic films, or fabrics. Suitable methods forimmobilizing peptides on solid phases include ionic, hydrophobic,covalent interactions and the like.

The methods of the present invention do not require the use of solidphases or substrates, however. The skilled artisan will recognize thatthere are a number of ways that the present method may be carried out todetect the presence or absence of roundworm without involving the use ofsolid phases or substrates. In just one example, immunoprecipitationmethods that do not require the use of solid phases or substrates may becarried out.

In some embodiments of the invention, the antigen/antibody complex isdetected when an indicator reagent, such as an enzyme conjugate, whichis bound to the antibody, catalyzes a detectable reaction. Optionally,an indicator reagent including a signal generating compound may beapplied to the antigen/antibody complex under conditions that allowformation of a detectable antigen/antibody/indicator complex.Optionally, the antibody may be labeled with an indicator reagent priorto the formation of an antigen/antibody complex.

The formation of an antigen/antibody complex or anantigen/antibody/indicator complex in some of the methods of the presentinvention specifically may be detected by radiometric, calorimetric,fluorometric, photometric, size-separation, or precipitation methods.Detection of an antigen/antibody complex also may be accomplished by theaddition of a secondary antibody that is coupled to an indicator reagentincluding a signal generating compound. Indicator reagents includingsignal generating compounds (labels) associated with apolypeptide/antibody complex may be detected using the methods describedabove and may include chromogenic agents, catalysts such as enzymeconjugates, fluorescent compounds such as fluorescein and rhodamine,chemiluminescent compounds such as dioxetanes, acridiniums,phenanthridiniums, ruthenium, and luminol, radioactive elements, directvisual labels, as well as cofactors, inhibitors, magnetic particles, andthe like. Examples of enzyme conjugates include alkaline phosphatase,horseradish peroxidase, beta-galactosidase, and the like. The selectionof a particular label is not critical, but it will be capable ofproducing a signal either by itself or in conjunction with one or moreadditional substances.

Methods of the invention include, but are not limited to those based oncompetition, direct reaction or sandwich-type assays, including, but notlimited to ELISA, RIA, immuno-fluorescent assays (IFA), hemagglutination(HA), fluorescence polarization immunoassay (FPIA), and microtiter plateassays (i.e., any assay done in one or more wells of a microtiterplate). One assay of the invention includes a reversible flowchromatographic binding assay, which may be performed, for example, byusing a SNAP® device. See U.S. Pat. No. 5,726,010.

In some embodiments, the method of the invention facilitates sandwich orcompetition-type specific binding assays. In a sandwich assay, antigencapture reagents are immobilized in a reactive zone. These antigencapture reagents may specifically bind to antigens in the sample beingtested for roundworm. Following binding of the antigen from the sample,the antigen capture reagent/antigen complex is detected by any suitablemethod. For example, the complex may be reacted with labeled specificbinding reagents (e.g., an enzyme-antibody conjugate) and antigendetected (e.g., upon reaction with substrate).

In other embodiments of the method of the present invention, acompetition assay is performed. In a competition assay, antigen capturereagents are immobilized at the reactive zone and are contactedsimultaneously with antigen from a sample and labeled antigen (e.g., anantigen-enzyme conjugate). The amount of label detected at the reactivezone is inversely proportional to the amount of antigen in the sample.

In some embodiments of the method, antibodies specific for a roundwormantigen or antigens are attached to a solid phase or substrate. A samplepotentially including an antigen from roundworm is added to thesubstrate. Antibodies that specifically bind roundworm are added. Theantibodies may be the same antibodies used on the solid phase or theymay be from a different source or species. Further, these antibodies maybe linked to an indicator reagent, such as an enzyme conjugate. Washsteps may be performed prior to each addition. A chromophore or enzymesubstrate may be added and color may be allowed to develop. The colorreaction may be stopped and the color may be quantified using, forexample, a spectrophotometer, and/or the color may be subjectivelyassessed by the human eye.

In other embodiments of the method, antibodies specific for a roundwormantigen or antigens are attached to a solid phase or substrate. A samplepotentially including a roundworm antigen is added to the substrate.Second anti-species antibodies that specifically bind antigens ofroundworms are added. These second antibodies are from a differentspecies than are the solid phase antibodies. Third anti-speciesantibodies that specifically bind the second antibodies and that do notspecifically bind the solid phase antibodies are added. The thirdantibodies may include an indicator reagent, such as an enzymeconjugate. Wash steps may be performed prior to each addition. Achromophore or enzyme substrate may added and color may be allowed todevelop. The color reaction may be stopped and the color may bequantified using, for example, a spectrophotometer, and/or the color maybe subjectively assessed by the human eye.

In a specific example, the method of the present invention is performedin conjunction with a device that is a lateral flow assay device byadding a prepared mammalian sample to a flow matrix of the device at afirst region (a sample application zone). The prepared sample is carriedin a fluid flow path by capillary action to a second region of the flowmatrix where a particulate label capable of binding and forming a firstcomplex with an antigen in the sample exists. The particulate label canbe, e.g., a colored latex particle, dye sol, or gold sol conjugated toan antibody specific for a roundworm antigen. The first complex iscarried to a third region of the flow matrix where an antibody thatspecifically binds a roundworm antigen is immobilized at a distinctlocation. A second complex is formed between the immobilized antibodyand the first complex. The particulate label that is part of the secondcomplex can be directly visualized by the human eye.

Roundworm antibody may be an immobilized antigen capture reagent in areaction zone (solid phase). A second antigen capture reagent, i.e., asecond roundworm antibody that has been conjugated to a label, eithermay be added to the sample before the sample is added to the device, orthe second antigen capture reagent can be incorporated into the device.For example, the labeled antigen capture reagent may be deposited anddried on a fluid flow path that provides fluid communication between asample application zone and the solid phase. Contact of the labeledantigen capture reagent with the test sample can result in dissolutionof the labeled antigen capture reagent.

In one embodiment of the method of the present invention, roundwormantigen is detected by ELISA. Specific examples of the ELISA method ofthe present invention is described in the Example section includedherein. Although the present invention is described with respect tothose specific ELISA methods, however, it is to be understood that thoseof ordinary skill in the art will recognize that alternative, additionalor substitute ELISA steps may be used without deviating from the basicgoal achieved through this method of the invention.

In another embodiment of the present invention, roundworm antigen isdetected by using a lateral flow device, such as a SNAP® device, forexample.

Further, the methods of the invention for detection of roundworminfection can be combined with other diagnostic assays to detect thepresence of other organisms or conditions. For example, assays of theinvention can be combined with reagents that detect one or morenon-roundworm worm fecal parasites, one or more non-worm fecalparasites, one or more viruses, one or more fungi, one or more bacteria,one or more blood-borne parasites or occult blood or a combinationthereof. By providing two or more unique binding sites in a single assaydevice (such as, for example, two unique spots on a SNAP® assay device),the present invention allows for detection of two or more organisms froma single sample. In one embodiment, there are three unique spots fordetection of past or present infection or infestation from threeorganisms (the spots being either antigen or antibody binding reagents)from a single sample (i.e., the same individual sample is exposed to thethree capture reagents on a single device). In yet another embodiment,there are four unique spots for detection of past or present infectionor infestation from four organisms (the spots being either antigen orantibody binding reagents) from a single sample (i.e., the sameindividual sample is exposed to the four capture reagents on a singledevice. It is to be understood, however, that the same device mayinclude more than four unique spots and/or allow for the detection ofmore than four organisms.

The reagents for the detection of one or more non-roundworm wormparasites, one or more non-worm parasites, one or more viruses, one ormore fungi, or one or more bacteria may be, for example, one or moreantibodies or one or more antigens recognized by antibodies specific forone or more non-roundworm worm parasites, one or more non-wormparasites, one or more viruses, one or more fungi, or one or morebacteria.

When a device of the present invention includes reagents for thespecific detection of hookworm and reagents for the specific detectionwhipworm, for example, in addition to the reagents for detectingroundworm, the method of the present invention may involve using thatdevice for the additional purpose or purposes of determining whether thesample that is being tested for roundworm also includes hookworm and/orwhipworm. In this arrangement, therefore, the method/device of thepresent invention would not only be able to specifically confirm thatroundworm is present in or absent from any particular test sample, butit would also be useful for specifically confirming that the sampleincludes or does not include any antigen of hookworm and/or any antigenof whipworm. The capability to specifically detect roundworm and one ormore other organisms by applying a single sample to the device of theinvention would be useful to the caregiver of the animal from which thesample under test was obtained. A caregiver who learns that a sampleincludes both roundworm and whipworm, but not hookworm, for example,could use that knowledge to treat the mammal from which the sample wastaken specifically for roundworm by administering to that mammal a drugoptimally effective against roundworm and a second drug optimallyeffective against whipworm. Absent such knowledge, the caregiver may,for example, otherwise treat the mammal with a drug that is optimallyeffective against only roundworm, only whipworm, or neither roundwormnor whipworm (in such cases, the mammal would be at risk of receivingsuboptimal treatment). In addition, humans who may come in contact withthe infested animal or its excretions may be advised to take precautionsagainst acquiring the parasite or parasites. In this context, it isimportant to determine the worm species with high specificity, as somehelminths, such as roundworms and hookworms, can cause significantdisease (e.g., larval migrans, severe enteritis or allergic reactions)in humans, while it is generally accepted that whipworm does not play azoonotic role of importance in humans.

The method further may optionally include using one or more nucleicacids from roundworm, including, but not limited to, the nucleic acidsof the present invention, to determine the presence or absence ofroundworm in a mammalian sample. Such use of these nucleic acids fordetermining the presence of roundworm may be carried out before, afteror concomitantly with the carrying out of any other aspects of themethod, including the detection of roundworm by antibody. Therefore, inone aspect, after roundworm is detected or not detected in a particularsample and the mammal from which the sample was obtained is diagnosed aseither having or not having a roundworm infection, the sample (or alater-obtained sample from the diagnosed mammal) may be tested for thepresence or absence of any one or more of the nucleic acids, includingany one or more nucleic acids of the invention. Anyone failing to detectroundworm in a particular mammal by using one or more nucleic acids(after the roundworm had been detected by using one or more antibodies)would need to take into consideration the possibility that theantibodies had detected roundworm antigen prior to the appearance ofdetectable roundworm nucleic acid in the sample. In such an instance,the mammal's caregiver may elect to ignore the observation that thenucleic acid had failed to detect the roundworm and proceed withtreating the mammal specifically for roundworm infection based on theobservation that the antibodies had in fact detected roundworm. Inanother aspect, the nucleic acids are used to determine the presence orabsence of roundworm in a particular mammal, and then the presence orabsence of roundworm is further evaluated by using the antibodies of thepresent invention. Detection of one or more roundworm nucleic acids maybe carried out by using any nucleic acid detection techniques known tothe skilled artisan. For example, such detection may be carried out byperforming a PCR-based technique, such as, but limited to, for example,a real-time PCR-based technique. Exemplary PCR-based techniques aredescribed in, e.g., PCR Protocols (Methods in Molecular Biology), 2^(nd)ed., Bartlett and Stirling, eds., Humana Press (2003); and Sambrook andRussell, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press (2001); each one of which is incorporated herein byreference in its entirety.

The present invention is specifically described with reference to sixExamples; however, it is not to be construed as being limited thereto.

EXAMPLES

[Unless otherwise indicated, the following materials and techniques wereused to generate data described in one or more of Examples 1-6 asdescribed below. Polyclonal antibody preparation. The polyclonalantibody “anti-DIV6716 pAB” (IgG) was raised in rabbit against apolypeptide having amino acid sequence corresponding to SEQ ID NO:5 andpurified from serum by using standard methods. Briefly, nucleotides 50through 427 of SEQ ID NO: 1 were cloned in-frame into a vector (D8223,which is a derivative of pUC19) to create the plasmid D8339.Specifically, the 129 amino acids of SEQ ID NO:5 that follow themethionine residue at the N-terminus of that sequence correspond to aportion of SEQ ID NO:3 and are encoded for by the cloned portion of SEQID NO:1. In the D8339 plasmid, the N-terminal methionine residue wasencoded for by vector sequence at the junction of that plasmid where thevector was ligated to the cloned sequence from SEQ ID NO: 1.

DNA sequence encoding SEQ ID NO:5 was then cleaved from the D8339plasmid by restriction exonuclease digestion (NdeI and BamHI) andpurified. This purified sequence was then ligated to linearizedexpression vector, pET28a, and the resulting circular construct(pTDX204::DIV6716) was transformed into BL21 (DE3) E. coli cells. (Thecomplete sequence of the insert was confirmed by DNA sequence analysis.)Expression of His-tagged fusion protein was induced by addition of 1 mMIPTG to cultures of the transformed E. coli. Recombinant protein wassolubilized in 6 M urea and purified by nickel affinity and ion exchangechromatography. (This recombinant protein is hereinafter is referred toas “rDIV6716”.)

After rDIV6716 was introduced into rabbits, anti-DIV6716 pAB waspurified from the plasma of the immunized rabbits by isolating IgGantibody by protein G affinity chromatography. The polyclonal antibodyanti-DIV6716 pAB was used in all six Examples described herein.

Infection and anti-helminth treatment of canine and feline animals.Parasitic nematode infection was effected by orally administering about150-300 larvated eggs of either roundworm (Toxocara), hookworm(Ancylostoma canium), or whipworm (Trichuris vulpis) to a healthy canineor feline. (Specifically, T. canis was the roundworm that wasadministered to canine and T. cati was the roundworm that wasadministered to feline.) For Example 2, fecal samples were collectedfrom canines known to be naturally infected with heartworm (Dirofilariaimmitis). Further, for Examples 4 and 6 only, canines were treated atpost-infection day 91 with Interceptor® (milbemycin oxime), which is ananthelmintic agent commercially available from Novartis Animal HealthInc. of Basel, Switzerland, or felines were treated at post-infectionday 56 with Drontal® (praziquantel/pyrantel pamoate), which is ananthelmintic agent commercially available from Bayer HealthCare, LLC ofShawnee Mission, Kans., according to the manufacturer's protocol. It iswell known by those of ordinary skill in the art that Interceptor® andDrontal® are effective for the removal of roundworms (and otherparasitic worms) from canines and felines, respectively, within 72 hoursafter treatment. Infection was confirmed by microscopic observation ofworm ova in fecal samples obtained from these host animals.

Canine and feline fecal sample preparation. Canine and feline animalsknown to be free of parasitic worm infection or to be infected with oneof either roundworm, hookworm, whipworm or heartworm provided the sourceof fecal samples. Samples (approximately 1 gram) from frozen,unpreserved canine or feline fecal samples were suspended in 4 ml ofdiluent solution (“diluent solution” is 0.05 M Tris base; 1 mM EDTA;0.45% Kathon; 16 mg/ml gentamicin sulfate; 0.05% Tween-20; 40% fetalbovine serum; 10% rabbit serum; and 5% mouse serum). The suspension wascentrifuged at 4000 rpm for 20 minutes to produce a first supernatant.The first supernatant was centrifuged at 12000 rpm for 5 minutes toproduce a second supernatant, which is referred to herein as “fecalextract”.

ELISA assays. Purified anti-DIV6716 pAB (100 μl/well for all Examples;10 μg/ml for each Example but Example 3, and 3 μg/ml for Example 3) wasimmobilized by physical adsorption on Immulon 1B 96-well platesovernight at 4° C. The plates were then blocked with 1% BSA in 0.1M TrispH 7.0 at 4° C. overnight, followed by drying at room temperature.Approximately 100 μl of fecal extract was added to each well and allowedto incubate at room temperature for one hour. The wells were then washedfive times with a PBS-Tween-20 solution according to standard methodsknown to those of ordinary skill in the art. In a separate reactionvessel, free anti-DIV6716 pAB was labeled with horseradish peroxidase(HRP) by using the crosslinker succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to create aconjugate, and 10 μg/ml of this conjugate was added to each well havingimmobilized anti-DIV6716 pAB. Following a 30-minute incubation period atroom temperature, unbound conjugate was washed from the wells by usingPBS-Tween-20 solution according to standard methods known to those ofordinary skill in the art. 50 μl of TMBLUE® peroxidase substrate(SeraCare Life Sciences, West Bridgewater, Mass.) was then added to eachwell and the plates were incubated for 10 minutes at room temperature.After stopping each enzymatic reaction with 0.1% sodium dodecyl sulfate(SDS) following the 10-minute incubation period, the optical density(OD) value of each well of the 96-well plate was measured at A650 bystandard spectrophotometric techniques by using an ELISA plate reader togenerate an “OD650 value” (or, more simply, an “OD value”) for eachwell. In this arrangement, the OD value obtained for any particular wellof the 96-well plate was directly proportional to the amount ofspecifically bound antigen present in the well.

Example 1

Anti-DIV6716 pAB specifically binds roundworm in fecal samples obtainedfrom roundworm-infected canines.

It was a goal of Example 1 to determine whether anti-DIV6716 pABspecifically binds roundworm coproantigen in canines. Measured OD valuesfor fecal samples obtained from individual canines that were known tohave a roundworm-infection are shown in FIG. 7. Specifically, thesesamples correspond to the roundworm-infected canines that are identifiedas “round+SPY”, “round+QKZ”, “round+KWZ”, “round+WHY” and “round+RYZ” inFIG. 7. In this Example, an OD value also was measured for wholeToxocara canis extract (“Whole Toxocara”; added to the plate at 1 μg/ml)and for rDIV6716 (added to the plate at 1 μg/ml), which served aspositive controls. (Specifically, the whole Toxocara extract wasprepared as described in U.S. patent application Ser. No. 11/763,592,assigned to the assignee of the present invention, which is incorporatedherein by reference in its entirety.) Further, an OD value was measuredfor each one of two fecal extracts obtained from two canines that didnot have a parasitic worm infection (“negTIY” and “negSVY”) and for asample that did not contain any fecal extract (“Diluent Only”). (Theselatter three samples served as negative controls.)

The measured OD value of the fecal extract obtained from the negTIY andnegSVY canines was 0.10 and 0.13, respectively, and the measured ODvalue of the diluent only sample was 0.05. (The average OD value forthese negative control samples therefore was 0.09.) The anti-DIV6716 pABtherefore was considered to not have specifically bound antigen in anyone of these negative control samples.

Conversely, the average of the measured OD values of the samplesobtained from the “round+SPY”, “round+QKZ”, “round+KWZ”, “round+WHY” and“round+RYZ” canines was 1.97, which was more than 21 times higher thanthe average OD value measured for the three negative control samples.These data indicate that anti-DIV6716 pAB specifically binds one or moreroundworm coproantigens.

Example 2

Anti-DIV6716 pAB specifically binds roundworm coproantigen, but does notspecifically bind coproantigen from either hookworm, whipworm orheartworm.

It was a goal of Example 2 to determine whether anti-DIV6716 pABspecifically binds coproantigen of hookworm and/or whipworm in canines.Measured OD values for pooled canine fecal extracts are shown in FIG. 8.Specifically, these fecal extracts were derived from fecal samplesobtained from five canine animals known to be infected with roundworm(“Roundworm-infected”), two canine animals known to be infected withhookworm (“Hookworm-infected”), and five canine animals known to beinfected with whipworm (“Whipworm-infected”). Additionally, OD valueswere also measured for whole Toxocara canis extract (“Whole Toxocara”; 1μg/ml) and rDIV6716 (1 μg/ml), which served as positive controls, andfor a pooled sample of fecal extracts obtained from five canines knownto be free of parasitic worm infection (“Uninfected”) and for a samplethat did not contain any fecal extract (“Diluent Only”). (These lattertwo samples served as negative controls.)

Referring to FIG. 8, the OD value measured for each one of thehookworm-infected and whipworm-infected samples was 0.09, whichapproximated or equaled the measured OD values of the negative controldiluent only sample (0.05) and the negative control uninfected sample(0.09). These data indicate that anti-DIV6716 pAB did not specificallybind coproantigen in either of the samples obtained from thehookworm-infected and whipworm-infected canines.

Conversely, the measured OD value of the pooled fecal extract from theroundworm-infected canines was 0.44, which was about five times higherthan obtained for the negative control samples and the hookworm-infectedand whipworm-infected samples. These data indicate that anti-DIV6716 pABspecifically binds one or more roundworm antigens, but does notspecifically bind any hookworm or whipworm coproantigen.

It was another goal of Example 2 to determine whether anti-DIV6716 pABspecifically binds coproantigen of heartworm in canines, as heartwormshave a close phylogentic relationship with roundworms, raising thepossibility of cross-reactivity. Measured OD values for individualcanine fecal extracts are shown in FIG. 9. Specifically, these sampleswere obtained from heartworm-infected canines that are identified as“TRS 405”, “TRS 583”, and “TRS 868” in FIG. 9. In this Example, an ODvalue also was measured for each one of two fecal extracts obtained fromtwo canines that did not have a parasitic worm infection (“negTIY” and“negSVY”). (These latter two extracts served as negative controls.)

Referring to FIG. 9, the average of the OD values measured for theheartworm-infected samples was 0.21 (with the largest of these OD valuesbeing 0.27), which approximated (and was actually less than) the averageof the two OD values measured for the negative control samples (0.33).These data indicate that anti-DIV6716 pAB did not specifically bind anycoproantigen in the samples obtained from the heartworm-infectedcanines.

Example 3

Anti-DIV6716 pAB specifically binds roundworm coproantigen, but does notspecifically bind coproantigen from either hookworm, whipworm orheartworm, and specific binding of roundworm coproantigen byanti-DIV6716 pAB produces a calorimetric change that is readilyobservable to the human eye.

It was a goal of Example 3 to determine whether specific binding betweenanti-DIV6716 pAB and roundworm coproantigen while the anti-DIV6716 pABis immobilized on a solid support can produce a calorimetric change thatis observable to the human eye.

Referring to FIG. 10, anti-DIV6716 pAB (3 μg/ml) was immobilized ontothe bottom surfaces of wells A1-A12 and B1-B12 of a microtiter plate asdescribed before. Following such immobilization, the A3 and B3 wellswere exposed to fecal extract from a heartworm-infected canine(indicated by “HW” in FIG. 10). The A4 and B4 wells were exposed tofecal extract from a first hookworm-infected canine, the A5 and B5 wellswere exposed to fecal extract from a second hookworm-infected canine,and the A6 and B6 wells were exposed to fecal extract from a thirdhookworm-infected canine. The A7 and B7 wells were exposed to fecalextract from a first roundworm-infected canine, the A8 and B8 wells wereexposed to fecal extract from a second roundworm-infected canine, andthe A9 and B9 wells were exposed to fecal extract from a thirdroundworm-infected canine. The A10 and B10 wells were exposed to fecalextract from a first whipworm-infected canine, the A11 and B11 wellswere exposed to fecal extract from a second whipworm-infected canine,and the A12 and B12 wells were exposed to fecal extract from a thirdwhipworm-infected canine. The A1 and B1 wells were exposed to rDIV6716(1 μg/ml), and therefore those wells served as positive controls. The A2and B2 wells were not exposed to any fecal extract or to rDIV6716, andtherefore those wells served as negative controls.

Following incubation of all of these wells with TMBLUE® peroxidasesubstrate and the subsequent addition of the SDS, colorimetric changewas visually observed in each one the wells that had been exposed tofecal extract from roundworm-infected canines (A7-A9 and B7-B9), but nocalorimetric change was observed in any of the wells that had beenexposed to fecal extract from canines infected with either hookworm,whipworm or heartworm.

These data indicate that anti-DIV6716 pAB detects roundworm sufficientlyenough to produce a colorimetric change that is robust and readilyvisible to the human eye. Further, these data indicate that suchcalorimetric change allows the human eye to readily distinguishroundworm-positive fecal samples from those that do not containroundworm, including those that include one or more of hookworm,whipworm, or heartworm.

Example 4

Anti-DIV6716 pAB detects roundworm coproantigen in some canines as earlyas 17 days after being infected with roundworm, and anti-DIV6716 pABdoes not detect roundworm in feces of canine animals that have had aroundworm infection, but that have been rid of that infection by thetime the feces were excreted by the canines.

It was a goal of Example 4 to determine whether anti-DIV6716 pAB candetect roundworm coproantigens in at least some roundworm-infectedcanines before roundworm ova first appear in the feces of those canines.It was another goal of Example 4 to determine whether anti-DIV6716 pABdetects roundworm in feces of canine animals that have been rid of aprior roundworm infection.

Toward these goals, OD values were measured for fecal samples obtainedfrom five canines and are shown in FIG. 11. These canines, which areidentified as “QKZ”, “RYZ”,“KWZ”, “SPY” and “WHY”, were infected withroundworm on day 0 and were treated with the Interceptor® anthelminticagent on day 91 after the administration of the infection as describedbefore. Fecal samples were taken from all or some of these canines onday 0, on day 2 and day 111 following the administration of theroundworm infections to these animals, and on selected days between day2 and day 111. Microscopic observation of the fecal samples from thefirst set of canines confirmed that each one of the samples taken at day0 through day 31 and at day 100 through day 111 was substantially freeof roundworm ova, and that, with one exception, such ova were presentonly in the samples at each one of days 38 through 97. (The loneexception being that ova were not observed in the day 38 fecal samplefrom the KWZ canine.)

Referring to FIG. 11, the average OD value measured for these fivecanines at day 17 was 0.35, which was five times higher than was theaverage of the OD values (0.07) that were measured for those canines atday 0. Further, the specific OD value that was measured for the WHYcanine at day 17 (which was 0.78) was more than 11 times higher than wasthe average of the OD values that were measured for all five canines atday 0, and the specific OD values that was measured for the WHY canineat days 23 and 31 (0.18 and 0.29, respectively) were more than two andmore than four times higher, respectively, than was the average of theOD values that were measured for all five canines at day 0. These dataindicate that, in some cases, anti-DIV6716 pAB can detect roundworm infeces from a roundworm-infected canine as early as 17 days after thecanine first became infected with roundworm.

With continuing reference to FIG. 11, the OD values measured for thefecal samples taken from the five canines at days 38 through 93 weremany times higher than were the OD values measured for fecal samplesfrom those same canines following their treatment with the anthelminticagent. These data indicate that anti-DIV6716 pAB does not detectroundworm in feces from a canine that has been rid of a prior roundworminfection.

Example 5

Anti-DIV6716 pAB specifically binds roundworm antigen in fecal samplesobtained from roundworm-infected feline animals.

It was a goal of Example 5 to determine whether anti-DIV6716 pABspecifically binds roundworm coproantigen in felines.

OD values measured for fecal samples obtained from uninfected felinesand roundworm-infected felines are shown in FIG. 12. Specifically, theseOD values were measured from fecal samples taken from five differentroundworm-infected felines (represented by the identifiers “C085”,“CO87”, “CO96”, “CO 100” and “CO 104”) 40 days following administrationof a roundworm infection to those felines. As a negative control, ODvalues also were measured from a fecal sample obtained from the C)85feline one day prior to the administration of the roundworm infection tothat feline (“day −1”).

Referring to FIG. 12, the OD value measured for the uninfected feline(CO85 at day −1) was 0.06, whereas the average OD value of the fivefelines at day 40 was 1.76. The average OD value measured from the fecalsamples obtained from the five felines at day 40 therefore was almost 30times greater than was the OD value measured for the uninfected feline.These data indicate that anti-DIV6716 pAB specifically binds one or moreroundworm coproantigens in feline.

Example 6

Anti-DIV6716 pAB does not detect roundworm in feces of feline animalsthat have had a roundworm infection, but that have been rid of thatinfection by the time the feces were excreted by the felines.

It was a goal of Example 6 to determine whether anti-DIV6716 pAB detectsroundworm in feces of feline animals that have been rid of a priorroundworm infection.

OD values measured for fecal samples obtained from a first set of sixfelines and a second set of six felines are shown in FIGS. 13 and 14,respectively. The first set of felines, which are identified as “C96”,“C100”,“C107”, “C85”, “C87” and “C104”, were infected with roundworm onday 0 and were treated with the Drontal® anthelmintic agent on day 56after the administration of the infection as described before. Fecalsamples were taken from all or some of the first set of felines on day0, day 1 and day 77 following the administration of the roundworminfections to these animals, and on selected days between day 1 and day77. Microscopic observation of the fecal samples from the first set offelines confirmed that each one of the samples taken at day 0 throughday 26 and at day 60 through day 77 was substantially free of roundwormova, and that such ova were present in each one of the day 34 throughday 54 samples.

The second set of felines, which are identified as “C91”, “C97”,“C106”,“C81”, “C98” and “C 118”, were never infected with roundworm (andtherefore served as negative controls). Fecal samples were taken fromeach one of these felines on the day that the first set of felines wereinfected with roundworm (day 0). Further, fecal sample were taken fromthese second set of felines on day 1 and day 74 following theadministration of the roundworm infections to the first set of felines,and on selected days between day 1 and day 74. Microscopic observationof the fecal samples from the second set of felines confirmed that eachone of the samples taken at day 0 through day 74 was free of roundwormova.

Referring to FIG. 13, the OD values measured for the fecal samples takenfrom the first set of felines (i.e., the felines that were infected withroundworm) at days 34 through 54 were many times higher than were the ODvalues measured for fecal sample samples from those same felinesfollowing their treatment with the anthelmintic agent. Further, the ODvalues measured for the fecal samples taken from the first set offelines at days 34 through 54 were many times higher than for each oneof the negative control samples of FIG. 14. These data further indicatethat anti-DIV6716 pAB does not detect roundworm in feces from a felinethat has been rid of a prior roundworm infection.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms, while retaining their ordinary meanings. The terms andexpressions which have been employed are used as terms of descriptionand not of limitation, and there is no intention in the use of suchterms and expressions of excluding any equivalents of the features shownand described or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments, optional features,modification and variation of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the description and the appended claims.

A number of examples to help illustrate the invention have beendescribed. Nevertheless, it will be understood that variousmodifications may be made without departing from the spirit and scope ofthe invention. Accordingly, other embodiments are within the scope ofthe claims appended hereto.

1. A method of detecting the presence or absence of one or moreroundworm antigens in a sample, the method comprising: (a) contacting asample from a mammal with one or more antibodies that can specificallybind to a polypeptide consisting of the amino acid sequence SEQ ID NO:5,SEQ ID NO:6; (b) forming antibody polypeptide complexes in the presenceof the polypeptide if any, in the sample; and (c) detecting the presenceor absence of the antibody-polypeptides complexes, if any.
 2. The methodof claim 1 further comprising contacting the sample with one or morereagents to detect one or more of the group consisting of: one or morenon-roundworm worm parasites, one or more non-worm parasites, one ormore viruses, one or more fungi, and one or more bacteria.
 3. The methodof claim 2 wherein the reagents for the detection of any one or all ofthe one or more non-roundworm worm parasites, one or more non-wormparasites, one or more viruses, one or more fungi and one or morebacteria are one or more antibodies or one or more antigens recognizedby antibodies specific for the one or more non-roundworm worm parasites,one or more non-worm parasites, one or more viruses, one or more fungior one or more bacteria.
 4. A method of diagnosing whether a mammal isinfected with an intestinal roundworm infection, the method comprisingthe steps of: (a) contacting a sample from a mammal with one or moreantibodies that can specifically bind to a polypeptide consisting of theamino acid sequence SEQ ID NO:5, SEQ ID NO:6; (b) formingantibody-polypeptide complexes in the presence of the polypeptides, ifany, in the sample; (c) detecting the presence or absence of theantibody-polypeptide complexes, if any; and (d) diagnosing the mammal ashaving the roundworm infection if a roundworm antibody-polypeptidecomplex is present.
 5. The method of claim 4 further comprisingcontacting the sample with one or more reagents to detect one or more ofthe group consisting of: one or more non-roundworm worm parasites, oneor more non-worm parasites, one or more viruses, one or more fungi, andone or more bacteria.
 6. The method of claim 5 wherein the reagents forthe detection of any one or all of the one or more non-roundworm wormparasites, one or more non-worm parasites, one or more viruses, one ormore fungi and the one or more bacteria are one or more antibodies orone or more antigens recognized by antibodies specific for one or morenon-roundworm worm parasites, one or more non-worm parasites, one ormore viruses, one or more fungi or one or more bacteria.
 7. The methodof any one of claim 1 or 4 wherein the sample is obtained from a mammalthat is a canine or a feline.
 8. The method of claim 7 wherein theroundworm is Toxocara canis or Toxocara cati.
 9. The method of any oneof claim 1 or 4 wherein the sample is a fecal sample.
 10. The method ofclaim 9 wherein the one or more antibodies do not specifically bind anycoproantigen derived from the group consisting of hookworm, whipworm,and heartworm.
 11. The method of any one of claim 1 or 4 wherein thestep of detecting the presence or absence of the complexes furtherincludes the step of providing at least one secondary antibody thatbinds to at least one of the complexes.
 12. The method of claim 11wherein the at least one secondary antibody is labeled.
 13. The methodof any one of claim 1 or 4 wherein one or more of the one or moreantibodies are labeled.
 14. The method of any one of claim 1 or 4wherein the one or more antibodies are immobilized on a solid support.15. The method of claim 14 wherein the solid support forms part of anenzyme-linked immunosorbent assay device.
 16. The method of claim 15wherein the enzyme-linked immunosorbent assay device is a lateral flowimmunoassay device.